Fragile X mental retardation syndrome is one of the most common human genetic diseases and the most common cause of hereditary mental retardation, affecting about 1 in 1200 males and 1 in 2500 females. Reviewed in Oostra, et al., (1993) in Genome analysis: Gemone mapping and neurological disorder, Vol. 6, K. E. Davies and S. M. Tilghman (Eds.), Cold Spring Harbor Laboratory Press, New York, pp. 45-75 and Nussbaum, R. L. and Ledbetter, D. H., (1995) in Metabolic Basis of Inherited Disease, C. R. Scriver, et al. (Eds.), McGraw Hill, New York, 7th Ed., pp. 795-810. The syndrome is characterized by mental retardation (average I.Q. of 20-60) and varying degrees of autistic behavior, macroorchidism in adult males, characteristic facial features and hyperextensible joints. Hagerman, R. J., (1991) in Fragile X syndrome: Diagnosis, treatment, and research, R. J. Hagerman and A. C. Silverman (Eds.), Johns Hopkins University Press, Baltimore, pp. 1-68.
The gene directly responsible for fragile X syndrome, FMR1, is located at Xq27.3. Verkerk, et al., Cell, 1991, 65, 905-914. The nucleotide and amino acid sequences of FMR1 are set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively. The 5' untranslated region of the FMR1 gene contains a polymorphic CGG trinucleotide repeat, 6-60 repeats found in normal individuals, which can be amplified to hundreds or thousands of copies in affected patients. Verkerk, et al., Cell, 1991, 65, 905-914; Oberle, et al., Science, 1991, 252, 1097-1102; Yu, et al., Science, 1991, 252, 1179-1181; Fu, et al., Cell, 1991, 67, 1047-1058. Fragile X syndrome usually results from expansion of the CGG repeats leading to hypermethylation of the CpG island adjacent to FMR1 and loss of transcription of the FMR1 gene. Pieretti, et al., Cell, 1991, 66, 817-822. Indeed, affected patients usually do not have detectable FMR1 protein. Siomi, et al., Cell, 1993, 74, 291-298 and Verheij, et al., Nature, 1993, 363, 722-724. The FMR1 mRNA and protein are expressed in many tissues, but particularly high levels are found in the brain and in tubules of the testes which are two of the major organs affected in fragile X syndrome. Hinds, et al., Nature Genet., 1993, 3, 36-43; Devys, et al., Nature Genet., 1993, 4, 335-340; Abitbol, et al., Nature Genet., 1993, 4, 147-152; and Bachner, et al., Nature Genet., 1993, 4, 115-116.
FMR1 knockout mice have been generated. These knockout mice lack normal FMR1 mRNA and protein expression and show enlarged testes, impaired cognitive function, and abnormal behavior. This animal model supports the central role of FMR1 in fragile X syndrome and it may serve as a valuable tool for the elucidation of the physiological role of FMR1. Bakker, et al., Cell, 1994, 78, 23-33.
The FMR1 protein contains motifs characteristic of RNA-binding proteins, namely two KH domains and an RGG box (Siomi, et al., Cell, 1993, 74, 291-298; Ashley, et al., Science, 1993, 262, 563-566; and Gibson, et al., Trends Biochem. Sci., 1993, 18, 331-333), and has been shown to bind RNA in vitro (Siomi, et al., Cell, 1993, 74, 291-298; and Ashley, et al., Science, 1993, 262, 563-566). Importantly, the RNA-binding activity of the FMR1 Ile-304.fwdarw.Asn mutant (which changes a highly conserved residue in the KH domain) that was found in a patient with severe fragile X syndrome (De Boulle, et al., Nature Genet., 1993, 3, 31-35), is impaired (Siomi, et al., Cell, 1994, 77, 33-39). Together, these findings suggest a strong connection between fragile X syndrome and the RNA-binding activity of FMR1. However, the cognate RNA target of FMR1 and its precise functions have not yet been elucidated.
PCT Publication WO 90/05194, which is incorporated herein by reference, describes a probe that is used to detect fragile X syndrome. The probe consists of a nucleic acid fragment that is hybridizable with the human X chromosome at the region Xq27.3-DXS52.
PCT Publication WO 91/09140, which is incorporated herein by reference, describes a probe for the detection of fragile X syndrome. The probe comprises at least 17 contiguous nucleotide bases.
PCT Publication WO 92/20825, which is incorporated herein by reference, describes nucleotide sequences and cosmids used to detect fragile X syndrome. The nucleotide sequences correspond to the FMR-1 gene.
PCT Publication WO 86/05512, which is incorporated herein by reference, describes DNA probes that recognize the polymorphic locus of the q28 region of the X chromosome.
PCT Publication WO 93/15225, which is incorporated herein by reference, describes a method of amplifying and detecting specific GC-rich nucleic acid sequences by polymerase chain reaction (PCR), which may be used to detect individuals with fragile X syndrome. The method determines whether the number of CGG repeats in the test individual's X-chromosome are characteristic of a normal, carrier, or afflicted person. Such a method is used to amplify and detect the GC-rich region of the FMR1 gene.
PCT Publication WO 92/12262, which is incorporated herein by reference, describes DNA sequences that may be used to detect individuals with fragile X syndrome. The DNA sequences span the fragile X site on the human X chromosome.
PCT Publication WO 92/14840, which is incorporated herein by reference, describes nucleic acid fragments containing mutations associated with the fragile X syndrome that may be used to detect individuals with mental retardation.
There remains a need for reagents, kits and methods useful in the identification of individuals suffering from fragile X syndrome. There is a need for reagents, kits and methods useful in the identification of individuals suffering from FMR1 deficiency without misdiagnosing an individual's condition due to cross-reactivity with fragile X related proteins.